ABSTRACT BACKGROUND/OBJECTIVE After digestion, dietary triacylglycerol stimulates incretin release in humans, mainly through generation of 2-monoacylglycerol, an agonist for the intestinal G

protein-coupled receptor 119 (GPR119). Enhanced incretin release may have beneficial metabolic effects. However, dietary fat may promote weight gain and should therefore be restricted in

obesity. We designed C4-dietary oil (1,3-di-butyryl-2-oleoyl glycerol) as a 2-oleoyl glycerol (2-OG)-generating fat type, which would stimulate incretin release to the same extent while

providing less calories than equimolar amounts of common triglycerides, e.g., olive oil. SUBJECTS AND METHODS We studied the effect over 180 min of (a) 19 g olive oil plus 200 g carrot, (b)

peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) responses (incremental area under curve (iAUC)): iAUCGLP−1: 645 ± 194 and 702 ± 97 pM × min; iAUCGIP: 4,338 ± 764 and

2,894 ± 601 pM × min) compared to the carrot meal (iAUCGLP−1: 7 ± 103 pM × min; iAUCGIP: 266 ± 234 pM × min). iAUC for GLP-1 and GIP were similar for C4-dietary oil and olive oil, although

olive oil resulted in a higher peak value for GIP than C4-dietary oil. CONCLUSION C4-dietary oil enhanced secretion of GLP-1 and GIP to almost the same extent as olive oil, in spite of

liberation of both 2-OG and oleic acid, which also may stimulate incretin secretion, from olive oil. Thus, C4-dietary oil is more effective as incretin releaser than olive oil per unit of

energy and may be useful for dietary intervention. SIMILAR CONTENT BEING VIEWED BY OTHERS COMPARISON OF PHARMACOKINETICS OF OMEGA-3 FATTY ACID SUPPLEMENTS IN MONOACYLGLYCEROL OR ETHYL ESTER

IN HUMANS: A RANDOMIZED CONTROLLED TRIAL Article Open access 03 October 2020 EGG AND SATURATED FAT CONTAINING BREAKFASTS HAVE NO ACUTE EFFECT ON ACUTE GLYCEMIC CONTROL IN HEALTHY ADULTS: A

RANDOMIZED PARTIAL CROSSOVER TRIAL Article Open access 09 November 2021 CALORIC RESTRICTION IMPROVES GLYCAEMIC CONTROL WITHOUT REDUCING PLASMA BRANCHED-CHAIN AMINO ACIDS OR KETO-ACIDS IN

OBESE MEN Article Open access 11 November 2022 INTRODUCTION Weight loss is an important goal in the treatment of obesity and diabetes, and can be achieved by a lower intake of dietary fat as

it is known that a higher dietary fat intake is associated with weight gain.1 Furthermore, the drug orlistat,2 which decreases fat absorption, as well as substitution of long-chain

triglycerides with medium-chain triglycerides in the diet3 are known to produce weight loss in humans. In a human weight loss regime, dietary fat restriction was, calorie for calorie, more

efficient than carbohydrate restriction with respect to body fat loss,4 supporting substitution of some of the fat in the human diet with carbohydrate or protein for prevention of weight

gain. However, dietary fat is a potent stimulus for incretin release,5 which appears to have several beneficial functions in energy metabolism.6 Upon digestion of dietary fat, it is

hydrolyzed to 2-monoacylglycerol and fatty acids, which both are absorbed in the small intestine, re-esterified and exported to the circulation with chylomicrons. It turns out that it is the

hydrolysis products that stimulate incretin release,7,8,9,10 although recent results suggest that fatty acids may stimulate glucagon-like peptide 1 (GLP-1) release from the vascular site

and not the luminal site.11 At a dose of dietary fat reflecting a light meal, e.g., 20 ml olive oil (21.6 mmol), it appears that it is mainly the 2-monoacylglycerol moiety of the fat that is

responsible for stimulating incretin release, whereas the fatty acid moieties may have a minor role.12 This conclusion was reached from studies of a C8-dietary oil, 1,3-di-octanoyl-2-oleoyl

glycerol, which upon digestion results in formation of two molecules of a medium-chain fatty acid, octanoic acid, and 2-oleoyl glycerol (2-OG). Octanoic acid is a poor agonist for fatty

acid receptors,13 and dietary medium-chain triglycerides are poor stimulators of gut hormone release,12, 14 while 2-OG alone readily stimulates release of incretins via activation of the G

protein-coupled receptor 119 (GPR119).12, 15, 16 Thus, on a molar basis the C8-dietary oil has the same potency as long-chain-triglycerides, although it provides only 61% of the calories,

i.e., for 21.6 mmol, 107 kcal versus 171 kcal.12 However, prolonged intake of octanoic acid may have the disadvantage of stimulating the formation of bioactive _N_-octanoyl-ghrelin,17, 18

which may promote food intake. Thus, we have chosen to study another oil in which octanoic acid is substituted by butyric acid in a similarly structured triacylglycerol, i.e.,

1,3-dibutyryl-2-oleoyl-glycerol, henceforth designated C4-dietary oil, which also contains even less calories (81 kcal for 21.6 mmol) on a molar basis. From mouse studies, butyric acid is

known to be able to elevate proglucagon gene expression in the intestine,19 increase the number of enteroendocrine L cells,20 improve vascular function and inhibit inflammation, probably via

inhibition of histone deacetylases involved in epigenetic regulations.21, 22 Furthermore, tributyrin/butyrate had anti-obesity and anti-diabetic effects after prolonged feeding in

mice23,24,25 as also seen with microbiota-derived short-chain fatty acids; the latter apparently exerted their anti-obesity effect via GPR43 activation.24 Targeted delivery of another

short-chain fatty acid, propionate, had beneficial effect on body weight maintenance and adiposity in overweight adults.26 Butyric acid was reported to stimulate GLP-1 release from murine L

cells,27 although an intake of 6.53 g tributyrin did not stimulate incretin release in humans.12 In the present study, we tested whether intake of 10.7 g C4-dietary oil (expected to generate

7.7 g 2-OG) stimulates incretin release in 13 overweight diabetic subjects. The results were compared to those obtained after intake of 19 g (20 ml) olive oil, which provides both

long-chain fatty acids and 2-OG (7.7 g equal to 21.6 mmol) and many more calories (177 versus 81 kcal) or compared to a carrot meal alone. MATERIAL AND METHODS The pro-drug, C4-dietary oil,

was first tested as an agonist as compared to the well-described agonist oleoylethanolamide (OEA)28 for transiently expressed human GPR119 in COS-7 cells, and subsequently in 13 patients

with type 2 diabetes (T2D) in a randomized, single-blinded study. We compared the effect of C4-dietary oil (active compound), olive oil (active comparator) on a background of grated carrot

(“placebo”) administered orally as three different “meals” in a cross-over design. The two oils were consumed in a shot-glass while eating the grated carrot. The primary outcome was

postprandial plasma responses of GLP-1 measured during and 3 h after ingestion of the different meals (assessed as incremental area under the curve (iAUC)). Secondly, we investigated the

effects of the meals on glucose-dependent insulinotropic polypeptide (GIP), on insulin and glucagon release from the pancreas, and on the appetite-regulating gut hormones peptide YY (PYY)

and cholecystokinin (CCK). IN VITRO STUDIES GPR119 SIGNALING COS-7 cells were grown at 10% CO2 and 37 °C (310 K) in Dulbecco’s modified Eagle’s medium supplemented with GlutaMAX™ (Life

Technologies Corporation, Carlsbad, CA, USA), 10% fetal bovine serum, 180 IU/ml penicillin and 45 µg/ml streptomycin (PenStrep) and were transfected by the calcium phosphate precipitation

method.29 The cAMP formation was determined by the HitHunter cAMP XS+assay (DiscoveRx, Fremont, CA, USA).30 In brief, the transiently transfected cells were seeded into 96-well plates

(35,000 cells/well). Twenty-four hours later, cells were washed twice with HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) and incubated with ligands (C4-dietary oil (Larodan, Malmø, Sweden)

or OEA (Cayman Chemicals, Ann Harbor, MI, USA)) for 30 min at 37 °C in HBS containing 1 mM isobutylmethylxanthine phosphodiesterase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). For the in

vitro experiment C4-dietary oil was dissolved in 50% DMSO and then diluted with medium to the desired concentrations, having a final concentration of DMSO of 1%. After incubation, the medium

was removed and the cells were treated according to the protocol for the “three reagent addition” procedure using the HitHunter cAMP XS+assay, an enzyme fragment complementation-based cAMP

assay. The amount of cAMP was measured as luminescence using Perkin Elmer EnVision 2104 Multilabel Reader (Walton, MA, US). Determinations were made in triplicate. IN VIVO STUDIES ETHICAL

APPROVAL The study was conducted according to the principles of the Helsinki Declaration II and approved by the Scientific-Ethical Committee of the Capital Region of Denmark (Registration

Number H-3-2011-007). SUBJECTS Thirteen overweight Caucasians patients (eight males, five females) diagnosed with T2D (according to World Health Organization criteria31) participated. All

patients were recruited from diabetes outpatient clinics in order to reflect clinical reality. Patient characteristics are listed in Table 1. All participants had negative tests of islet

cell auto-antibodies and glutamate decarboxylase 65 auto-antibodies. Exclusion criteria included kidney disease with serum creatinine >130 µM and/or albuminuria, liver disease with plasma

alanine transaminase >twice the upper limit of the normal range, diabetic neuropathy, proliferative retinopathy, anemia, treatment with insulin, GLP-1 receptor agonist and/or dipeptidyl

peptidase 4 (DPP-4) inhibitor and inability to pause medication for at least 10 h. The subjects had a ‘low’ HbA1c but they paused their antidiabetic medication (biguanide or sulfonylurea) 7

days prior to each test day and therefore present with a fairly high fasting blood glucose level. All subjects agreed to participate by signing an informed consent after receiving oral and

written information prior to procedures. The participants were identified by a study protocol identification number. STUDY DESIGN The study was designed as a randomized, single-blinded

cross-over study. It was blinded for the patients and analyst but it was not possible to blind it for the staff handling the different oils. The subjects were studied in a recumbent position

after an overnight (10 h) fast including water, medication and tobacco abstinence. A cannula was inserted into a cubital vein. The cannulated arm was wrapped in a heating pad throughout the

experiment for collection of arterialized blood samples. On three different days and in random order the participants received the different test meals, consisting of 200 g grated carrot

and 1.0 g acetaminophen dissolved in 40 ml water plus a shot glass of either (a) 19 g of olive oil, or (b) 10.7 g of diet oil, or (c) no addition. Acetaminophen was added in order to measure

gastric emptying rate. Two-hundred grams carrot was used as vehicle since it had no measurable effect on incretin hormone concentrations in peripheral plasma.12 There were 2–10 days between

the different study days in order to avoid too much baseline variation. Theoretically, both oil “meals” would result in formation of 7.7 g 2-OG during digestion. The caloric content of the

oils was 171 kcal (olive oil) and 81 kcal (C4-dietary oil), respectively, calculated on the basis of the amount of ATP generated from total oxidation of the two oils, and the notion that

dietary fat contains 9 kcal/g. As shown in the figures arterialized blood was drawn at specific timepoints after ingestion of the meal and dispensed into chilled tubes containing EDTA plus

aprotinin (500 KIU/ml blood; Trasylol®, Bayer Corp., Leverkusen, Germany) and a specific DPP-4 inhibitor (valine-pyrrolidide (0.01 mM); a gift from Novo Nordisk, Bagsværd, Denmark) for

analysis of glucagon, GIP, GLP-1, PYY, CCK and triglycerides. For insulin and C-peptide analyses, blood was sampled into chilled tubes containing heparin. All tubes were kept on ice before

and after blood sampling. They were centrifuged for 20 min at 1.500_g_ and 4 °C. Plasma for glucagon, GLP-1, GIP, PYY, CCK and triglyceride analyses was stored at −20 °C and plasma for

insulin and C-peptide analyses was stored at −80 °C until analysis. For analysis of acetaminophen, blood was distributed into dry vials and left to coagulate for 20 min at room temperature

before centrifugation and handling as described below. For bedside measurement of plasma glucose (PG), blood was collected in fluoride tubes and centrifuged (7.400_g_) immediately for 2 min

at room temperature. SOLUTIONS Olive oil (Frantoio 100% italiano, non filtrato, olio extra vergine di oliva) was from the manufacturer “Rocchi” (Oleificio R.MSant’Alessio Lucca, Italy).

C4-dietary oil (1,3-dibutyryl 2-oleoyl glycerol of > 97% purity, with impurities being mainly 1,2-dibutyryl 3-oleoyl glycerol) was from Larodan (Malmø, Sweden). 1,3-dibutyryl 2-oleoyl

glycerol is a natural constituent of milk fat.32 ANALYSES Plasma samples were assayed for total GLP-1 immunoreactivity, as previously described,33 using antiserum no. 89390, which is

specific for the C-terminal of the GLP-1 molecule. Intact, biologically active GIP was measured using antiserum no. 98171 as previously described.34 PYY(3–36) was measured using a

commercially available radioimmunoassay kit from Millipore (catalog no. PYY-67HK; Billerica, MA, USA). The detection limit was 5 pM. The assay shows no cross-reaction with PYY(1–36) up to

250 pM. All quality controls were within prespecified limits. The concentrations of PG were measured bedside by the glucose oxidase method using a glucose analyzer (YSI model 2300 STAT plus

analyzer; Yellow Springs Instrument Inc., Yellow Springs, OH, USA). Plasma C-peptide and insulin concentrations were measured using two-sided, electrochemiluminescense immunoassays

(Roche/Hitachi modular analytics; Roche Diagnostic GmbH, Mannheim, Germany). The detection limit was less than 2 pM for both assays, and intra-assay coefficients of variation were 4.6% for

the C-peptide assay and 1.9% for the insulin assay.35 The glucagon assay is directed against the C terminal of the glucagon molecule (antibody code no. 4305) and therefore measures glucagon

of mainly pancreatic origin.36,37,38 CCK was measured as described earlier using antiserum no. 92128, which binds all bioactive forms of CCK and displays no cross-reactivity with gastrin.39

Triglycerides were measured using an enzymatic kit from Sigma-Aldrich (catalog no. TR0100; Saint Louis, MO, USA) with a modified protocol using 2 µl of plasma. Enzymatic reactions were

performed in duplicates and measured in 96-well plates at 550 nm. Triglyceride concentrations were calculated according to the protocol. The working range was from 0.7 mM to 11.3 mM defined

by a recovery of at least 80% and a coefficient of variation of <10%. Serum samples were assayed for acetaminophen using Vitros Chemistry Systems (Otho Clinical Diagnostics, Johnson &

Johnson, Buckinghamshire, UK). STATISTICAL ANALYSES AND CALCULATIONS All results are expressed as means ± SEM unless otherwise mentioned. Statistical analysis was performed using Prism

Graphpad 4 software (GraphPad Software, Inc., CA, USA). Repeated measurements ANOVA was followed by Tukey post hoc test. Area under curve (AUC) values were calculated using the trapezoidal

rule and are presented as the incremental (area below baseline subtracted) AUC (iAUC) values if nothing else is stated in order to adjust for different baseline values. Furthermore, we

analyzed differences in individual time points using paired _t_ tests. The acetaminophen absorption test was used to determine gastric emptying rates40 calculated from tAUC. RESULTS IN VITRO

DATA The study on the COS-7 cells shows as expected no effect of C4-dietary oil in vitro (Fig. 1), in contrast to the potent stimulation by OEA with a half maximal effective concentration

(EC50) of 1.2 × 10−7 M. IN VIVO DATA Fourteen participants were initially recruited of whom one did not complete the study. This participant was excluded the 1st day of the study, because he

was unable to eat 200 g carrot and vomited during ingestion of C4-dietary oil. GLP-1, GIP AND PYY Baseline plasma values for GLP-1 (olive oil: 10.0 ± 0.9 pM, C4-dietary oil: 8.1 ± 0.5 pM

and carrot: 8.7 ± 0.6 pM. _P_ = 0.01) and GIP (olive oil: 12.5 ± 2.1 pM, C4-dietary oil: 7.3 ± 1.2 pM, carrot: 8.6 ± 1.0 pM. _P_ = 0.02), respectively, differed slightly on the different

study days, while there was no difference for PYY (Table 2). Ingestion of both olive oil and C4-dietary oil in combination with carrot elicited greater GLP-1 (olive oil: 645 ± 194 pM × min,

C4-dietary oil: 702 ± 97 pM × min, carrot: 7 ± 103 pM × min. _P_ = 0.002) and GIP (olive oil: 4338 ± 764 pM × min, C4-dietary oil: 2894 ± 601 pM × min, carrot: 266 ± 234 pM × min. _P_ < 

0.0001) responses compared to the meal consisting of only carrot, and there were no differences in iAUC between olive oil-day and C4-dietary oil-day (Table 3). The peak value for GLP-1 was

not different between the oil-days, whereas the peak value for GIP was higher for the olive oil-day than for the C4-dietary oil-day (olive oil: 68.85 ± 9.24 pM, C4-dietary oil: 38.92 ± 7.10 

pM, carrot: 19.54 ± 2.46 pM. _P_ < 0.0001 (Table 4)). iAUC and peak values for PYY were similar on the 3 days (Fig. 2, Table 3). GLUCOSE, INSULIN, C-PEPTIDE AND GLUCAGON There were no

differences in baseline concentrations or iAUCs for C-peptide or glucose responses, respectively (Fig. 2, Table 3). Insulin iAUC was greater with C4-dietary oil compared to carrot alone

(olive oil: 8438 ± 2222 pM × min, C4-dietary oil: 10,690 ± 1671 pM × min, carrot: 4901 ± 1883 pM × min. _P_ = 0.02 (Table 3)). Baseline concentrations of glucagon were similar during the

three study days. A very slight but higher glucagon response was observed for C4-dietary oil compared to olive oil and grated carrot (olive oil: 17 ± 104 pM × min, C4-dietary oil: 420 ± 95 

pM × min, carrot: 18 ± 83 pM × min. _P_ = 0.005 (Table 3)). CCK AND TRIGLYCERIDES No differences in baseline concentrations were observed between the three experimental days for CCK or

triglycerides, respectively (Table 2). Olive oil elicited a greater rise in triglyceride concentrations whereas C4-dietary oil and carrots were without effect (olive oil: 12.0 ± 6.8 mM × 

min, C4-dietary oil: −4.4 ± 3.9 mM × min, carrot: −7.3 ± 6.0 mM × min. _P_ = 0.013 (Table 3, Fig. 2)). A higher CCK response was seen following olive oil compared to grated carrot (olive

oil: 103 ± 34 pM × min, C4-dietary oil: 33 ± 37 pM × min, carrot: −4 ± 22 pM × min. _P_ < 0.01 (Table 3, Fig. 2)). There was no difference for iAUCCCK between olive oil and C4-dietary

oil, and both elicited peak values that were higher than the control meal with only carrot (olive oil: 2.71 ± 0.36 pM, C4-dietary oil: 2.73 ± 0.40 pM, carrot: 1.37 ± 0.16 pM. _P_ = 0.0040

(Fig. 2, Table 3, Table 4)). GASTRIC EMPTYING RATE Gastric emptying rate estimated as the tAUC for acetaminophen, was lower with olive oil compared to the carrot meal (olive oil: 11.7 ± 1.0 

µM × min, C4-dietary oil: 13.4 ± 1.2 µM × min, carrot: 14.0 ± 1.2 µM × min. _P_ = 0.02 (Table 3)). DISCUSSION Our in vitro data demonstrate that C4-dietary oil by itself cannot activate

GPR119, in accordance with the observation that a hydroxyl group in the lipid molecule is required for agonist activity on GPR119.15 This indicates that C4-dietary oil, like other

triglycerides, must be hydrolyzed before it can activate nutrient sensors.41, 42 Several different lipids (lysophopholipids, acylamides, 2-monoacylglycerols) can act as agonists for GPR119

in vitro.15, 43 We have shown that 2-OG stimulates GPR119 not only in vitro, but probably also in vivo where direct administration of 2 g 2-OG in the intestine of healthy subjects resulted

in an increase in plasma GLP-1 within 25 min after administration.16 Oleic acid in an amount corresponding to that present in 2 g 2-OG did not stimulate GLP-1 release, suggesting that the

meal-related formation of 2-monoacylglycerols acting on GPR119 during fat ingestion is indeed responsible for a significant part of the GLP-1 response. Recently, we have shown that compared

to 20 ml (19 g) olive oil, an equimolar amount of 1,3-dioctanoyl 2-oleoyl glycerol (13.15 g, 21.6 mmol) was just as effective with respect to GLP-1 release in humans, suggesting that the

stimulating factor in the consumed olive oil may be mainly 2-OG acting on GPR119.12 Studies of a mouse model involving self-administration of fat emulsions also demonstrated the mice can

sense 2-OG in the gastrointestinal system probably via GPR119 activation.44 The present in vivo study was designed to characterize the effect of C4-dietary oil on incretin hormone secretion

compared with equimolar amounts of olive oil and with grated carrots, respectively, in overweight patients with T2D. By examining the same individuals in a cross-over design inter-individual

variation was minimized. Patients were naturally more well treated due to exclusion criteria, but they were recruited from diabetes outpatient clinics in order to reflect reality well.

Although fatty acids generated from the digestion of olive oil may stimulate incretin release via GPR40 and GPR1207, 8 or other mechanisms,9 they are also rapidly absorbed together with

2-monoacylglycerol and subsequently re-esterified. Apparently, at a dose of 20 ml olive oil to healthy human subjects, the long-chain fatty acid moiety of olive oil seems to play a minor

role in GLP-1 release, while it seems to have an additive effect together with 2-OG on GIP release and be of major responsibility for CCK release.12 In the present study, C4-dietary oil also

gave the same GLP-1 response as olive oil, further supporting the GLP-1-releasing action of 2-OG, while the C4 fatty acid (butyrate) seems to have no effect, as judged from the finding that

an oral intake of an equimolar amount of tributyrin did not stimulate GLP-1 release.12 The iAUCs for GIP on the olive oil-day and the C4-dietary oil day were not different although the peak

value for GIP was clearly higher on the olive oil-day. In our previous study, we found that the GIP response to olive oil can be explained by a combined stimulatory effect of both 2-OG and

oleic acid,12 and the present data tend to support this conclusion. Since the incretin responses to 19 g of olive oil (171 kcal) and only 10.7 g of C4-dietary oil (81 kcal) were similar,

this indicates that C4-dietary oil, per unit of energy content, is more effective than olive oil in releasing GLP-1 and probably also GIP. Neither olive oil nor C4-dietary oil had effects on

iAUC for PYY, although our previous study demonstrated a clear PYY-stimulating effect of equimolar doses of both C8-dietary oil and olive oil in healthy subjects.12 Whether this difference

in PYY responses is due to testing the oils on overweight patients with T2D as compared to the previous study, where they were tested on healthy subjects is at present not clear. However,

Fernandez-Garcia et al.45 has shown that in morbidly obese subjects, both insulin resistance and abnormal glucose metabolism impair GLP-1 and PYY responses to a 60 g fat load. Also, the 2-OG

receptor GPR119 has been reported to be involved in stimulating PPY release.46 In the present study, both oils stimulated CCK release with olive oil being more efficacious than C4-dietary

oil, while in our previous study C8-dietary oil and tributyrin did not stimulate a CCK response.12 One explanation for these differences in CCK responses between the C8-dietary oil study12

and the present C4-dietary oil study may possibly be related to the rate of gastric metabolism of these two structured triglycerides. Due to the short-chain fatty acids in C4-dietary oil it

may have been metabolized more rapidly in the stomach by gastric lipase than C8-dietary oil. This would result in the generation of 2-OG having longer time to undergo spontaneous acyl

migration47 before entering the intestine, thereby resulting in formation of 1-oleoyl glycerol, which is substrate for hydrolysis by both gastric lipase and pancreatic lipase.48 This would

result in a larger proportion of free oleic acid generated from C4-dietary oil than from C8-dietary oil. Long-chain fatty acids are known to stimulate CCK release.49, 50 GLP-1 secretion may

be stimulated by CCK.51 However, in our study we saw a GLP-1 release in the C4-dietary oil group after 75 min when CCK levels were low. In earlier studies, we did not observe increased GLP-1

secretion after CCK administration, however,52 so in support of our earlier studies12, 16 it seems that a GLP-1 release induced by 2-OG can be elicited without involving CCK release.

Administration of all three meals was followed by an increase in the PG (no difference among meals) reflecting digestion of the carbohydrates in carrots.12 There was a tendency that the oil

meals elicited higher C-peptide responses (Fig. 2). However, the greater increase in both GLP-1 and GIP following ingestion of oils compared to carrots was not sufficient to influence

insulin secretion in these experiments. GLP-1 is known to increase insulin secretion in a dose-dependent manner.53,54,55 In a previous study, GLP-1 infusions resulting in peripheral plasma

GLP-1 concentrations of ∼50 pM stimulated insulin secretion at fasting glucose levels, but in the current study, the increase of GLP-1 from 8 to 15 pM might be too small to influence insulin

secretion significantly.53 We know from previous studies55 that in order to demonstrate the insulinotropic effect of GLP-1 and GIP at fasting PG concentrations, it is necessary to clamp PG

most thoroughly. That was unfortunately not done in the current study. We also know that portal insulin levels can be up to six times higher than the peripheral levels given that the flow of

the splanchnic circulation is one third of the systemic and that the hepatic insulin extraction is 50%. Therefore, we cannot say that the incretin release was insufficient in this study. On

the contrary, these data suggest an effect due to the relatively low glucose values on oil days. In animal studies in which GPR119 agonists were administered in high doses without

influencing glucose concentrations,56 stimulation of the GPR119 also had no insulinotropic activity at lower glucose concentrations. In addition, the lack of acute effect on insulin

secretion in our study may be due to the decreased sensitivity of the beta cells to incretin hormones in T2D, as shown by Højberg et al.57 In that study, no increase in insulin release

following intravenous infusion of GLP-1 and GIP (resulting in physiological plasma concentrations), respectively, was observed. Gastric emptying might have influenced the results observed in

this study. Our results show that olive oil inhibited gastric emptying. In our previous study,12 we saw no clear effect of the same dose of 20 ml olive oil on gastric emptying in healthy

subjects. Gastric emptying rate was evaluated using the acetaminophen absorption test. The test is an indirect assessment of gastric emptying, and it is well validated against scintigraphic

methods considered the gold standard for measurements of gastric emptying.58, 59 In the present study, there were no differences in gastric emptying between C4-dietary oil and carrot alone,

suggesting that fatty acids from olive oil may contribute to the effect on gastric emptying in these overweight patients with T2D. Recently, it has been shown that diabetic mice lacking the

short-chain receptors, GPR41 and GPR43, have increased insulin secretion and improved glucose tolerance60 thereby questioning the beneficial metabolic role of dietary short chain fatty acids

via this pathway, although prolonged oral intake of butyrate in diet-induced obese mice resulted in increased insulin sensitivity and increased energy expenditure.23 Anyhow, both C4-dietary

oil and C8-dietary oil increased GLP-1 release just as efficaciously as equimolar amounts of olive oil, which provided more calories. Further studies evaluating chronic administration of

C4-dietary oil or C8-dietary oil for a longer period should therefore be carried out, since beta cell sensitivity to incretin hormones may improve during anti-diabetic therapy.49 In

conclusion, our study suggests that postprandial GLP-1 and GIP responses in overweight patients with T2D may be augmented by GPR119 activation. C4-dietary oil had on a molar basis the same

activity as olive oil. However, if one calculates by unit of energy, C4-dietary oil may be more potent as incretin releaser than olive oil and could possibly be useful for some dietary

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FFA3 increases insulin secretion and improves glucose tolerance in type 2 diabetes. _Nat. Med._ 21, 173–177 (2015). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are

grateful to H. Frandsen helping recruiting patients and to H. Lethmar, A. Jans, M. R. Andersen, L. Albæk and S. Pilgaard for technical assistance. The work was supported by The P. Carl

Petersen Foundation, The Johannes Fog Foundation, The Augustinus Foundation and UNIK (Food, Fitness & Pharma for Health and Diseases), which is supported by the Danish Ministry of

Science, Technology and Innovation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * The Novo Nordisk Foundation Center for Basal Metabolic Research, Faculty of Health and Medical Sciences,

University of Copenhagen, Copenhagen, Denmark Mette Johannsen Mandøe, Katrine Bagge Hansen, Johanne Agerlin Windeløv, Filip Krag Knop & Jens Juul Holst * Department of Biomedical

Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Mette Johannsen Mandøe, Katrine Bagge Hansen, Johanne Agerlin Windeløv & Jens Juul Holst *

Department of Clinical Physiology and Nuclear Medicine, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark Mette Johannsen Mandøe & Katrine Bagge Hansen * Center for

Diabetes Research, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark Filip Krag Knop * Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of

Copenhagen, Copenhagen, Denmark Filip Krag Knop * Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Jens Frederik Rehfeld * Department of

Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Mette Marie Rosenkilde * Department of Drug Design and Pharmacology,

Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Harald Severin Hansen Authors * Mette Johannsen Mandøe View author publications You can also search for

this author inPubMed Google Scholar * Katrine Bagge Hansen View author publications You can also search for this author inPubMed Google Scholar * Johanne Agerlin Windeløv View author

publications You can also search for this author inPubMed Google Scholar * Filip Krag Knop View author publications You can also search for this author inPubMed Google Scholar * Jens

Frederik Rehfeld View author publications You can also search for this author inPubMed Google Scholar * Mette Marie Rosenkilde View author publications You can also search for this author

inPubMed Google Scholar * Jens Juul Holst View author publications You can also search for this author inPubMed Google Scholar * Harald Severin Hansen View author publications You can also

search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Jens Juul Holst. ETHICS DECLARATIONS CONFLICTS OF INTEREST The authors declare that they have no

conflict of interest. ADDITIONAL INFORMATION Clinical trial ID: NCT01453842. URL: http://clinicaltrials.gov/show/NCT01453842 RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under

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license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Mandøe, M.J., Hansen, K.B., Windeløv, J.A. _et al._ Comparing olive

oil and C4-dietary oil, a prodrug for the GPR119 agonist, 2-oleoyl glycerol, less energy intake of the latter is needed to stimulate incretin hormone secretion in overweight subjects with

type 2 diabetes. _Nutr & Diabetes_ 8, 2 (2018). https://doi.org/10.1038/s41387-017-0011-z Download citation * Received: 14 October 2016 * Revised: 01 August 2017 * Accepted: 06 November

2017 * Published: 12 January 2018 * DOI: https://doi.org/10.1038/s41387-017-0011-z SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get

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